1. Field of the Invention
The present invention relates to methods for the noninvasive detection of colonic biomarkers using fecal messenger RNA (mRNA). More particularly, the present invention relates to methods for the isolation of poly A+ RNA from feces, and includes the subsequent detection of, and quantitation of, particular mRNAs that correlate with a patient's diagnosis and/or prognosis of colon cancer thereby providing methods for noninvasively diagnosing and/or prognosticating colon cancer in a patient. One embodiment of the present invention relates to the detection of, and quantitation of, mRNA from sloughed colon cells in feces encoding particular isozymes of protein kinase C (PKC) whose levels are correlative with and predictive of colon cancer in a patient. Methods including semi-quantitative RT-PCR and biochip microarray technology may be made to assay and evaluate the fecal poly A+RNA.
2. General Background
Since colon cancer is the second most common cause of U.S. cancer deaths and since early detection can result in a high cure rate, an accurate screening method for colon cancer is imperative. Current detection methods have many drawbacks. For example, fecal occult blood screening can produce false positive results due to meat consumption, iron supplement intake and other common behaviors. The other routine screening technique, sigmoidoscopy, is an invasive expensive procedure which has inherent risks of perforation, reaction to sedative, or bleeding. In addition, the efficacy of sigmoidoscopy screening remains unproven (Levin, 1996). Because of these limitations, colon cancer cure rates have not improved in the past 30 year (Silverberg, 1988, WFR/AICR, 1997). Therefore, an accurate technique to detect early changes associated with the tumorigenic process is imperative in order to decrease the mortality from colon cancer.
Screening of colorectal cancer is recommended for all persons aged 50 and older with annual fecal occult blood testing or sigmoidoscopy, or both (Levin, 1996). However, each of these tests has limitations related to sensitivity and specificity (Levin, 1996). The presence of colorectal and pancreatic tumors has been detected in the stool and colonic effluent of patients by noninvasive methods based on the molecular pathogenesis of the disease (Sidransky, 1992: Tobi, 1994; Caldas, 1994). These protocols utilize DNA extraction procedures and the detection of oncogene mutations using PCR. The major disadvantage of this methodology is that it will not detect alterations in gene expression. Our methodology can quantitate the expression of any relevant gene by isolating and amplifying mRNA derived from fecal material containing sloughed colonocytes.
A sensitive molecular technique for the detection of colon cancer is important since early diagnosis can substantially reduce mortality (Levin, 1996). Our method is noninvasive, highly sensitive and specific. Our protocol is unique because it will determine colonic expression of any gene (e.g., tumor suppressor gene, oncogene), and provides early sensitive prognostic information and greatly enhances current methods of dietary and pharmacologic risk assessment.